L’Oréal on faster, animal-free allergen testing: Inside its one-hour probe

L’Oréal scientists have developed a faster, more reliable method for identifying cosmetic ingredients that may cause allergic reactions, without animal testing. The creation of the new probe molecule comes amid evolving cosmetics regulations that increasingly ban the use of animals in testing. Amid the regulatory shifts, companies are looking for faster and clearer lab assays to efficiently screen cosmetic raw ingredients.

According to the scientists, existing non-animal tests for skin sensitization require long incubation times and struggle to distinguish between direct and indirect sensitizers. Direct sensitizers, or electrophiles, react with the skin immediately, whereas indirect sensitizers, or pro-electrophiles, only become reactive after oxidation.

The scientists refer to this as a “gap” in current testing methods. They developed N,N-dimethyl N-(2-(1-naphthyl)acetyl)-L-cysteine, or NNDNAC, to fill that gap. The tool is a novel in chemico nucleophilic reactivity probe that rapidly assesses the skin sensitization potential of cosmetic ingredients.

NNDNAC can detect strong direct sensitizers within one hour, whereas established assays typically need a full 24-hour incubation period to reach the same level of detection.

“The difference between getting reliable results in just one hour versus 24 hours is truly significant for both the cosmetics industry and the broader scientific research community. It’s not just about saving time; it creates a ripple effect of benefits across the entire product development and research cycle,” Dr. Ratnadeep Paul Choudhury, head of analytical chemistry at L’Oréal India and contributing scientist to the study, tells Personal Care Insights.

“It allows for more experiments, faster decisions, lower costs, and ultimately, a more dynamic and responsive approach to ensuring chemical safety and driving innovation,” he continues.

Missing puzzle piece 

The development of NNDNAC aligns with the global shift toward New Approach Methodologies (NAMs) and the broader movement to replace animal testing in cosmetic safety assessment.

“This movement is driven by ethical considerations and regulatory bans on animal testing for cosmetics, particularly in regions like the EU,” Choudhury explains.

Regulations increasingly restrict animal testing, and the industry is turning to non-animal methods to assess skin sensitization.The ECHA has updated its guidance on how companies can meet EU REACH skin sensitization requirements without animal testing. Because no single test is sufficient on its own, the ECHA guideline instructs companies to combine several methods to determine whether a chemical is a skin sensitizer and how strong it is.

Skin sensitization is understood through the Adverse Outcome Pathway (AOP) framework, which outlines the molecular and cellular events leading to sensitization. The tool works by addressing Key Event 1 (KE1) of the AOP. 

KE1 is the molecular initiating event. It involves the covalent binding of electrophilic substances to nucleophilic centers in skin proteins.

“NNDNAC specifically targets and probes this initial event, making it a valuable tool within AOP-based assessment strategies,” says Choudhury.

“No single alternative method can fully replace in vivo evaluation. Therefore, the scientific community relies on [combined data] from various sources — including in chemico, in vitro, and in silico methods. NNDNAC provides critical, high-quality in chemico data for KE1, which can be integrated with other NAMs to build comprehensive, animal-free safety assessments.”

Differentiating hazards

NNDNAC is a molecule that behaves like the part of a skin protein that allergens usually react with. The researchers designed it so that this molecule is extra reactive — meaning it grabs onto sensitizing chemicals much faster than the probes used in standard tests.

According to the researchers, current established in chemico methods have limitations that impact their efficiency and mechanistic insight. Traditional assays often struggle to distinguish between direct sensitizers and pro-electrophiles.

Direct electrophiles are inherently reactive and immediately bind to skin proteins, triggering a potential allergic reaction. “Knowing a chemical is a direct electrophile tells us it’s a primary concern,” says Choudhury.

Chemical allergen safety testing is essential for ensuring that cosmetic ingredients do not trigger unwanted immune reactions.“Pro-electrophiles are more like a ‘sleeping giant.’ In their original form, they might not be harmful, but they transform into reactive sensitizers after being exposed to oxidation or undergoing metabolic changes in the body,” he explains.

If scientists can determine whether a chemical becomes reactive after prolonged air exposure or after being metabolized, their understanding of its true hazard (under typical product use) becomes more refined.

“Understanding this difference provides crucial mechanistic insight into how a substance causes sensitization, which is far more valuable than simply knowing if it causes it.”

Older tests might activate pro-electrophiles during the test itself, leading to a pro-electrophile being mistakenly classified as a direct and immediate sensitizer. “NNDNAC, by showing no reaction at one hour for pro-electrophiles, helps prevent over-classification,” says Choudhury.

The scientist tweaked the molecular structure of the skin protein to make the “reactive end” more eager to bond with allergens. In other words, the molecule is electronically boosted so it responds almost instantly, which is why the test works in one hour instead of a full day.

The distinction also helps formulators make more informed choices about which ingredients to use. A direct sensitizer could be avoided altogether, while a pro-electrophile could be used safely with appropriate formulation and stabilization strategies.

“The more detailed and accurate the information about a chemical’s reactivity, the more robust and reliable our overall animal-free safety assessment becomes. This is key to developing safe cosmetics without relying on animal testing,” Choudhury explains.

Breaking bottlenecks

Current screening processes for cosmetic ingredients are slow, which poses a challenge for rapid product development. The researchers envision that NNDNAC will help address this and contribute to faster lead times.

Faster testing tools are crucial for reducing development timelines and bringing safe products to market more efficiently.“When a researcher discovers or synthesizes a promising new raw material — perhaps a novel antioxidant from a plant extract or a new synthetic peptide — they no longer have to wait days or weeks for initial sensitization data. They can perform an NNDNAC test, get reliable results within an hour, and quickly decide if the ingredient is worth pursuing further or if it presents an immediate sensitization red flag,” explains Choudhury.

This speed and reliability help prioritize resources and avoid investing time and money in inherently problematic compounds.

The probe also aligns with the “Safe by Design” principle. “If we know a chemical is a pro-electrophile, we might be able to take steps to prevent its activation in a product. For example, we could add antioxidants to a formula to inhibit oxidation or choose packaging that minimizes air exposure. This allows for the safe use of ingredients that might otherwise be discarded,” Choudhury says.

Moreover, the tool is useful for quick troubleshooting. If a slight change in a manufacturing process or a raw material comes under scrutiny, NNDNAC can be deployed for rapid “what-if’ testing to see if the change introduces any new sensitization potential.

If a sensitizer then needs to be replaced, NNDNAC allows for the quick screening of numerous alternative compounds, thereby significantly speeding up the search for a safe and effective substitute.

“NNDNAC moves skin sensitization assessment from a potential bottleneck to a fluid, integrated part of the daily development process. It empowers researchers and formulators to make faster, more informed decisions, leading to quicker innovation, more efficient resource allocation, and ultimately, the delivery of safer, high-quality cosmetic products to consumers,” Choudhury concludes.